Fig. 4. Hh signaling promotes Sxl nuclear entry but does not require Smo. All discs were treated with 75-200 ng/ml LMB in DS2 medium for 3 hours before fixing. (A-C) hh^MRT disc stained for full-length Ci (A), Sxl (B), and merged image of A and B (C). Full-length Ci identifies areas in the disc with ectopic Hh expression. Sxl levels are highest where Ci is highest (e.g. arrow) and where Ci is lower (arrowhead) nuclear Sxl levels are also lower. Under these conditions the posterior compartment shows lower levels of nuclear Sxl (signal less bright) relative to the anterior compartment. (D-F) Enlargement of region that spans across high and low nuclear Sxl levels as well as full-length Ci, near the arrow in A-C. In F the region of higher Sxl signal has brighter green spots, which most probably reflect more nuclear Sxl than Ci. (G-I) hh^ts2 disc shifted to 29°C for 12 hours stained for Ci (G), Sxl (H) and merged image (I). The arrowhead in G marks the remnants of full-length Ci at the AP boundary and in H where the nuclear Sxl in the wing pouch is absent and the signal appears very diffuse. After 36 hours at 29°C, the low levels of nuclear Sxl in (H) appear reduced (not shown). (J-L) smo² clones in wing disc pouch at room temperature stained for Sxl (green), ß-gal (red) and (L) merged image of J and K. smo² clones in wing disc at 18°C stained for (M) ß-gal (red), (N) Sxl (green) and (O) with Hoechst (blue). Mutant clones marked by loss of ß-gal signal. There is no correspondence between the clones that mark the loss of Smo with the nuclear levels of Sxl (N). Clones (M), Sxl signal (N) and merged image (P). In N,O and Q where the optical section does not go through nuclei the Sxl signal is present but weaker as there is protein in the cytoplasm (marked by arrowhead). When the Sxl signal overlaps with the nuclear stain, a change in color to a lighter shade of blue is observed (Q). Line in J-Q marks the AP border. All are confocal images; anterior is to the left, ventral at the top. Scale bar: 40 µm in A-C; 20 µm in J-Q.

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